9419027 Gennaro, Maria During initiation of replication, the specific interaction between replication initiation protein(s) and the origin (ori)is often modulated by accessory factors that bind to specific DNA sequences called replication enhancers. The replication enhancer found in the staphylococcal plasmid pT181 has been identified and characterized during the previous granting period. The pT181 enhancer, designated cmp, stimulates the interaction of ori with the replication initiation protein, RepC, from a site located at a distance of 1,000 base pairs (bp) from ori. cmp binds a chromosomally-encoded accessory protein called CBF (cmp-binding factor). Dr. Gennaro was recently cloned the gene encoding CBF. These studies will advance the present understanding of a distant replication enhancer and its cognate accessory factor(s) and contribute to the definition of general rules underlying enhancement of DNA replication, such as involvement of accessory proteins, competition between origins in the same cell, and action at a distance. To achieve the goals, genetic and biochemical studies will be performed, often in parallel. Using the cloned cbf gene, the role of CBF in replication enhancement by cmp will be assessed a) by producing a host mutant carrying a defective cbf gene in which cmp activity can be studied in the absence of the CBF product, and b) by correlating in vivo activity of mutant cmp sequences with their ability to bind CBF in vitro. Three different models of replication enhancement by cmp are proposed. One model postulates a truly distant action by cmp on ori, while others involve protein-protein interactions between RepC and CBF. Since cmp activity is associated to changes in linking deficit (supercoiling), the nature of cmp-associated supercoiling changes will be determined by using torsionally tuned Z-DNA probes to measure intracellular superhelical tension in cmp+ and cmp- plasmids. Results of these supercoiling experiments should contribute support to one of the models pr oposed for cmp activity. Finally, a mutant screen will be undertaken, searching for host-linked mutations that suppress Cmp-. Putative host factor(s) obtained using this mutant screen should help define the intracellular signals to which cmp may respond to stimulate plasmid replication. Understanding the mechanism of action of accessory DNA sites located far from the origin of DNA replication and the soluble factors interacting with those sites is expected to contribute to our understanding of the regulatory circuitry governing initiation of DNA replication. %%% The overall goal of these studies is to elucidate the molecular mechanisms that regulate DNA replication. DNA replication is a key event in the cell cycle that determines efficient and faithful duplication of the genetic material. A variety of regulatory circuits are imposed upon the replication initiation step. Initiation can respond to stimulatory mechanisms that activate the origin, either transcriptionally or through the activity of distinct, often distant, DNA elements called replication enhancers. A major objective of these studies is to characterize the function of a bacterial replication enhancer and of the soluble protein factor(s) interacting with it. These studies are expected to advance our understanding of the complex regulatory circuitry governing cell growth and, of how different replicons are controlled within the same cell. *** ??
